Glycated hemoglobin is a generic term referring to a series of minor hemoglobin components that are formed via the attachment of various sugars, most commonly glucose, to the hemoglobin molecule. The most important of these minor hemoglobin components in respect to diabetes is hemoglobin A.sub.1c. It is formed by the attachment of glucose to the N-terminal amino acid residue, valine, on one or both .beta. chains of hemoglobin A (Goldstein, D. E., et al., Clin. Chem. 32:B64-B70, 1986).
The human erythrocyte is freely permeable to glucose. Within each erythrocyte, glycated hemoglobin is formed from hemoglobin A (the native, normal form) at a rate proportional to the ambient glucose concentration. The reaction is spontaneous, not enzyme catalyzed, but slow enough that only a fraction of the hemoglobin is modified during the life span of the erythrocyte (120 days) and is irreversible. As a result, glycated hemoglobin provides a weighted "moving" average measure of past blood glucose levels with more recent glucose levels having a greater influence (Singer, et al., Ann. Clin. Biochem. 26:213-219, 1989).
Elevated levels of glycated hemoglobin are known to be associated with diabetes mellitus. Glycated hemoglobin is present in non-diabetics at a level of about 5% of total hemoglobin, while diabetics have 2-4 times that amount. Glycated hemoglobin levels are relatively unaffected by short-term (hour-to-hour) fluctuations in blood sugar level and, hence give a relatively precise reflection of the state of blood glucose control in diabetics. The results are indicative of the time-average blood glucose concentration over the past 1 to 3 months. Glycated hemoglobin measurements are used in the assessment of the severity of glucose intolerance in a diabetic patient and in management of diabetes mellitus (Lester, Ann. Clin. Biochem. 26:213-219, 1989; Kennedy, et al., Br. Med. Bull. 45:174-190, 1989; Fluckiger, et al., J. Chromatogr. 429:279-292, 1988: Goldstein, et al., Clin. Chem. 32:B64-70, 1986; Mortensen, Dan. Med. Bull. 32:309-328, 1985; Goldstein, et al., CRC Crit. Rev. Clin. Lab. Sci. 21:187-228, 1984; Peacock, J. Clin. Pathol. 37:841-851, 1984; Miedema, et al., Ann. Clin. Biochem. 21:2-15, 1984; Mayer, et al., Clin. Chem. Acta 127:147-184, 1983; Gabbay, Med. Clin. North Am. 66:1309-1315, 1982).
There are various methods for measuring glycated hemoglobin; as hemoglobin A.sub.1c or hemoglobin A1, or as total glycated hemoglobin (ion-exchange chromatography, thiobarbituric acid method, isoelectric focusing, and affinity chromatography assays) (Cole, R. A., et al., Metabolism 27:289-301, 1978; Nathan, D. M., Clin. Chem. 27:1261-1263, 1981; Moore, J. C., et al., Ann. Clin. Biochem. 23:85-91, 1986). In ion-exchange chromatography many glycated hemoglobin species, including hemoglobin A.sub.1c, are less positively charged at neutral pH than hemoglobin A.sub.o, and bind less well to a negatively charged resin (Rosenthal, P. K., et al., AM. J. Clin. Pathol. 75:45-49, 1981; U.S. Pat. Nos. 4,407,961, 4,649,122). A few methods have been described that separate hemoglobin A.sub.1c from hemoglobin A.sub.1a+b fraction (Goldstein, D. E., et al., Diabetes 31:70-78, 1982; Maquart, F. X., et al., Clin. Chim. Acta 108:329-332, 1980; Jones, M. D., et al., Hemoglobin 2:53-58; 1978; Clarke, J. T., et al., Diabete Metabol. 5:293-296, 1979; Davis, J. E., et al., Diabetes 27:102-107, 1978; Cole, R. A., et al., Metabolism 27:289-301, 1978; U.S. Pat. No. 4,389,491; Bio-Rad Laboratories, Hemoglobin A.sub.1c Micro Column Test Instruction Manual, March 1990). However, these methods suffer from one or more disadvantages. Many of the methods involve the use of two buffers, the first to elute nonbound material from the ionexchange resin in such a way that does not cause the desorption of the specifically bound material. A second buffer, used at a different pH, ionic strength or containing a competitive inhibitor is needed to elute the specifically bound material. The temperature, pH, ionic strength or the presence of a competitive inhibitor is needed to elute the specifically bound material. The temperature, pH, ionic strength, and column size affect the test results (Simon, M., et al., Diabetes 29:467-474, 1980; Schellekens, A. P. M., et al., Clin. Chem. 27:94-99, 1981; Castagnola, M., et al., J. Chromatogr. 272:51-65, 1983). Moreover, the methods require several different steps, several vessels, and most of the methods are nonautomated or only semiautomated.
Other limitations to these assays, depending on the method used, include a reversible intermediate glycated form, "pre-hemoglobin-Alc", which needs to be removed before the assay is done (Goldstein, D. E., et al., Diabetes 31:70-78, 1982; Bunn, H. F., Diabetes 30:613-617, 1981; Nathan, D. M., Clin. Chem. 27:1261-1263, 1981; Mayer, T. K., et al., Clin. Chim. Acta 127:147-184, 1983; Health and Public Policy Committee, American College of Physicians Ann. Intern Med. 101:710-713, 1984) (Nathan, D. M. Clin. Chem. 27:1261-1263, 1981). High levels of fetal hemoglobin, sickle hemoglobin, and other rarer conditions may interfere with the assay (Niejadlik, D. C., et al., JAMA 224:1734-1736, 1973).
Other methods of determining glycated hemoglobin use specific affinity or binding agents to bind glycated hemoglobin. In the following patents, U.S. Pat. Nos. 4,200,435; 4,260,516; 4,274,978; 4,255,385, and 4,438,204, glycated hemoglobin is determined using affinity methods or the allosteric properties of hemoglobin. In DE Patent 1595 69, a sugar-binding protein as an affinity reagent is described.
Other affinity binding methods are based on specific complex formation between glycated hemoglobin and boronic acid derivatives (Middle, et al., Biochem. J. 209:771-779, 1983; Klenk, et al., Clin. Chem. 28:2088-2094, 1982; Little, et al., Clin. Chem. 32:358-360, 1986, U.S. Pat. Nos. 4,269,605; 4,861,728; UK Patent Application GB 2 206 411 A; Isolab, Inc. Technical Publication:Glyc-Affin.TM. GHb, 1986; Forrest, R. D., et al., Clin. Chem. 34:145-148, 1988). Although affinity binding methods detect glycated hemoglobin species in addition to HbA.sub.1c, they correlate linearly with methods more specific for HbA.sub.1c, such as ion-exchange chromatography (Little, et al., Clin. Chem. 32:358-360, 1986). Like the ion-exchange and colorimetric assay for glycated hemoglobin, the affinity methods also have limitations. One of the limitations is that two different buffers are required. The first buffer elutes the non-glycated fraction, which does not have cis-diol groups. The bound fraction, rich in glycated hemoglobin is eluted with a second buffer which contains a displacing agent, such as a sugar alcohol, that displaces glycated hemoglobin from the column. Additionally, the flow rate and size of the column limits the amount of hemoglobin bound to the affinity agent.
There is a need for a glycated hemoglobin assay that is easy to perform, free from interferences and relatively insensitive to experimental variables such as pH and temperature. An object of the present invention is to develop an assay method and reagents to perform glycated hemoglobin measurements accurately and with precision.